Background: Genotyping by sequencing, a new low-cost, high-throughput sequencing technology was used to genotype 2,815 maize inbred accessions, preserved mostly at the National Plant Germplasm System in the USA. The collection includes inbred lines from breeding programs all over the world. Results: The method produced 681,257 single-nucleotide polymorphism (SNP) markers distributed across the entire genome, with the ability to detect rare alleles at high confidence levels. More than half of the SNPs in the collection are rare. Although most rare alleles have been incorporated into public temperate breeding programs, only a modest amount of the available diversity is present in the commercial germplasm. Analysis of genetic distances shows population stratification, including a small number of large clusters centered on key lines. Nevertheless, an average fixation index of 0.06 indicates moderate differentiation between the three major maize subpopulations. Linkage disequilibrium (LD) decays very rapidly, but the extent of LD is highly dependent on the particular group of germplasm and region of the genome. The utility of these data for performing genome-wide association studies was tested with two simply inherited traits and one complex trait. We identified trait associations at SNPs very close to known candidate genes for kernel color, sweet corn, and flowering time; however, results suggest that more SNPs are needed to better explore the genetic architecture of complex traits. Conclusions: The genotypic information described here allows this publicly available panel to be exploited by researchers facing the challenges of sustainable agriculture through better knowledge of the nature of genetic diversity.